There are many types of the organic compounds known as indoles, and a variety of indoles are important as biological compounds and as pigments. Toxic indoles are present in some organisms.
The blue green algal organism Microcoleus lyngbyaceus is found in marine, brackish, and fresh water environments throughout the world. This species of algae is commonly encountered in the U.S.A., Canada, the Bahamas, the U.S. Virgin Islands, Bermuda, Haiti, Cuba, Jamaica, Mexico, Panama, Brazil, Argentina, Antarctica, Kenya, Libya, Egypt, Spain, Portugal, France, the Netherlands, England, Ireland, Scotland, Sweden, Norway, Russia, Denmark, Germany, Italy, Greece, Yugoslavia, Ethiopia, Union of South Africa, Malaysia, Indonesia, Vietnam, Australia, New Zealand, the Philippines, Taiwan, the Peoples Republic of China, Japan, Tonga, the Marquesas, French Polynesia, the Marshall Islands, and many other locations. In the U.S.A., Microcoleus lyngbyaceus has been reported in the states of Hawaii, Florida, Washington, California, Texas, Louisiana, Mississippi, Alabama, Georgia, South Carolina, North Carolina, Virginia, Maryland, Delaware, New York, Connecticut, Rhode Island, Massachusetts, Maine, and many other states.
In the State of Hawaii, the occurrence of Microcoleus lyngbyaceus has been reported for the islands of Kauai, Niihau, Oahu, Molokai, Lanai, Maui, and Hawaii. Toxic varieties of Microcoleus lyngbyaceus have been reported for Hawaii (e.g. specimens from the islands of Kauai, Oahu, Molokai, Maui, and Hawaii) and Ceylon (now Sri Lanka).
Toxic strains of the blue-green alga (i.e. phylum Cyanophyta) Microcoleus lyngbyaceus (formerly classified as Lyngbya majuscula) have been recognized as the etiologic agent of "stinging seaweed" dermatitis, a contact dermatitis, since the late 1950's and "stinging seaweed" escharotic stomatitis since 1979. Microcoleus lyngbyaceus dermatitis is characterized by burning sensations, itching, stinging, erythema, papules, vesicles, blisters, swelling (i.e., edema), denuded erosions, scalded appearance, eschar formation, scarring (variable), and lymphadenopathy (variable), generally in the bathing suit area, and/or conjunctivitis and rash, swelling, and pustular folliculitis over exposed parts of the body in some.
Human patch testing, animal patch testing, force feeding of animals and the investigation of natural outbreaks of human dermatitis attributable to Microcoleus lyngbyaceus have been some of the primary biological methods utilized in the identification of toxic versus non-toxic algal organisms. Chemical determination of algal toxicity became possible upon the extraction and subsequent identification of aplysiatoxin (Serdula, M. et. al., Hawaii Med. J., 41:200-201, 1982), debromoaplysiatoxin (Mynderse, J. S. et al., Science, 196:538-540, 1977), and lyngbyatoxin A (Cardellina, J. H. II et al., Science, 204:193-195, 1979), three irritating vesicants or blister producing agents from Microcoleus lyngbyaceus. As purified chemicals they produce irritation, blisters, and cutaneous pustules, and all three are suspected cocarcinogens (i.e., tumor promoters) (Nakayasu, M. et al., Cancer Letters, 12:271-277, 1981; Fujiki, H. et al., Proc. Natl. Acad. Sci. U.S.A., 78: 3872-3876, 1981).
The identification of one of these vesicants, lyngbyatoxin A, as an indole suggested that a histological staining procedure might be able to be utilized in order to qualitatively detect this toxin or derivative(s) of the toxin. The fact that lyngbyatoxin A gave a positive Ehrlich test for indoles (Cardellina, J. H. II et al., Science, 204:193-195, 1979) supported this approach. It was felt to be desirable to develop a histological technique which could be utilized on a routine basis by histopathology laboratories, hospitals or otherwise, particularly including application to tissues in paraffin blocks handled in a routine fashion. The development of a histological test or a chemical test was preferable to human patch testing or animal testing for considerations of potential carcinogenicity in both systems and cost in the latter testing program. The indole moiety has been detected by using the histochemical stain of Adams (Adams, C. W. M., J. Clin. Path., 10:56-62, 1957) hereby incorporated by reference. Ehrlich's reagent, or p-dimethylaminobenzaldehyde, which is used for the biochemical detection of indole derivatives, was reportedly utilized as a component to stain tissues for certain types of indoles. However, fading or loss of color can occur using this method.
The initial staining method utilized was the p-dimethylaminobenzaldehyde nitrite for tryptophane and related compounds (Adams, C. W. M., J. Clin. Path., 10:56-62, 1957); and Theory and Practice of Histological Techniques, Ed. J. D. Bancroft and A. Stevens, New York: Churchill Livingstone, 1977, p. 115). The p-dimethylaminobenzaldehyde, or DMAB, histochemical staining method for indoles is relatively specific for 2- or 3-open or reactive indoles (e.g., tryptophan, or serotonin) in regard to a blue reaction product (Adams, C. W. M., J. Clin. Path., 10:56-62, 1957). Other amino acids (e.g., histidine, tyrosine, cysteine, proline, arginine, lysine, et al., as tested (C. W. M. J. Clin. Path., 10:56-62, 1957), phenol (N.B., aplysiatoxin and debromoaplysiatoxin possess phenol moieties), quinone, phloroglucinol, resorcinol, aniline, benzidine, pyrrole, uric acid, and other substances tested (Adams, C. W. M., J. Clin. Path., 10:56-62, 1957) did not provide a blue coloration upon DMAB staining.
It is an object of the subject invention to provide a method for identifying certain indoles in biological materials.
A further object of the invention is to provide a relatively permanent biological stain for certain types of indoles.